The Sodium-Glucose Co-Transporter 2 (SGLT2) Inhibitor Empagliflozin Reverses Hyperglycemia-Induced Monocyte and Endothelial Dysfunction Primarily through Glucose Transport-Independent but Redox-Dependent Mechanisms.

PURPOSE: Hyperglycaemia-induced oxidative stress and inflammation contribute to vascular cell dysfunction and subsequent cardiovascular events in T2DM. Selective sodium-glucose co-transporter-2 (SGLT-2) inhibitor empagliflozin significantly improves cardiovascular mortality in T2DM patients (EMPA-REG trial). Since SGLT-2 is known to be expressed on cells other than the kidney cells, we investigated the potential ability of empagliflozin to regulate glucose transport and alleviate hyperglycaemia-induced dysfunction of these cells. METHODS: Primary human monocytes were isolated from the peripheral blood of T2DM patients and healthy individuals. Primary human umbilical vein endothelial cells (HUVECs) and primary human coronary artery endothelial cells (HCAECs), and fetoplacental endothelial cells (HPECs) were used as the EC model cells. Cells were exposed to hyperglycaemic conditions in vitro in 40 ng/mL or 100 ng/mL empagliflozin. The expression levels of the relevant molecules were analysed by RT-qPCR and confirmed by FACS. Glucose uptake assays were carried out with a fluorescent derivative of glucose, 2-NBDG. Reactive oxygen species (ROS) accumulation was measured using the H2DFFDA method. Monocyte and endothelial cell chemotaxis were measured using modified Boyden chamber assays. RESULTS: Both primary human monocytes and endothelial cells express SGLT-2. Hyperglycaemic conditions did not significantly alter the SGLT-2 levels in monocytes and ECs in vitro or in T2DM conditions. Glucose uptake assays carried out in the presence of GLUT inhibitors revealed that SGLT-2 inhibition very mildly, but not significantly, suppressed glucose uptake by monocytes and endothelial cells. However, we detected the significant suppression of hyperglycaemia-induced ROS accumulation in monocytes and ECs when empagliflozin was used to inhibit SGLT-2 function. Hyperglycaemic monocytes and endothelial cells readily exhibited impaired chemotaxis behaviour. The co-treatment with empagliflozin reversed the PlGF-1 resistance phenotype of hyperglycaemic monocytes. Similarly, the blunted VEGF-A responses of hyperglycaemic ECs were also restored by empagliflozin, which could be attributed to the restoration of the VEGFR-2 receptor levels on the EC surface. The induction of oxidative stress completely recapitulated most of the aberrant phenotypes exhibited by hyperglycaemic monocytes and endothelial cells, and a general antioxidant N-acetyl-L-cysteine (NAC) was able to mimic the effects of empagliflozin. CONCLUSIONS: This study provides data indicating the beneficial role of empagliflozin in reversing hyperglycaemia-induced vascular cell dysfunction. Even though both monocytes and endothelial cells express functional SGLT-2, SGLT-2 is not the primary glucose transporter in these cells. Therefore, it seems likely that empagliflozin does not directly prevent hyperglycaemia-mediated enhanced glucotoxicity in these cells by inhibiting glucose uptake. We identified the reduction of oxidative stress by empagliflozin as a primary reason for the improved function of monocytes and endothelial cells in hyperglycaemic conditions. In conclusion, empagliflozin reverses vascular cell dysfunction independent of glucose transport but could partially contribute to its beneficial cardiovascular effects.

Hyperglycemia-induced endothelial dysfunction is alleviated by thioredoxin mimetic peptides through the restoration of VEGFR-2-induced responses and improved cell survival.

BACKGROUND: Diabetes mellitus is an important cardiovascular risk factor characterized by elevated plasma glucose levels. High glucose (HG) negatively influences endothelial cell (EC) function, which is characterized by the inability of ECs to respond to vascular endothelial growth factor (VEGF-A) stimulation. We aimed to identify potential strategies to improve EC function in diabetes. METHODS AND RESULTS: Human umbilical cord endothelial cells (HUVECs) were subjected to hyperglycemic milieu by exposing cells to HG together with glucose metabolite, methylglyoxal (MG) in vitro. Hyperglycemic cells showed reduced chemotactic responses towards VEGF-A as revealed by Boyden chamber migration assays, indicating the development of "VEGF resistance" phenotype. Furthermore, HG/MG-exposed cells were defective in their general migratory and proliferative responses and were in a pro-apoptotic state. Mechanistically, the exposure to HG/MG resulted in reactive oxygen species (ROS) accumulation which is secondary to the impairment of thioredoxin (Trx) activity in these cells. Pharmacological and genetic targeting of Trx recapitulated VEGF resistance. Functional supplementation of Trx using thioredoxin mimetic peptides (TMP) reversed the HG/MG-induced ROS generation, improved the migration, proliferation, survival and restored VEGF-A-induced chemotaxis and sprouting angiogenesis of hyperglycemic ECs. Importantly, TMP treatment reduced ROS accumulation and improved VEGF-A responses of placental arterial endothelial cells isolated from gestational diabetes mellitus patients. CONCLUSIONS: Our findings suggest a putative role for Trx in modulating EC function and its functional impairment in HG conditions contribute to EC dysfunction. Supplementation of TMP could be used as a novel strategy to improve endothelial cell function in diabetes.